The recently identified family of RGS proteins plays a key role in the termination of signal transduction through the heterotrimeric G proteins. RGS act as GTPase-activating proteins (GAPs) for the G protein alpha subunits. This extremely high GAP activity in vitro implies that the signaling transmitted by G proteins can be terminated prematurely; therefore, researchers postulated that the activity of RGS proteins themselves should be regulated in vivo. However, the mechanisms of RGS regulation are currently now known. This proposal is based on our recent discovery that RGS proteins RGS6, 7 and 9 can directly interaction, in vivo and in vitro, with the G protein beta subunit Gbeta5. Our preliminary data demonstrate that Gbeta5 prevents the binding of RGS to Galpha, indicating that Gbeta5 which is significantly different from the four other Gbeta subunits in its primary structure and properties. In contrast to other Gbeta's which are always associated with a Ggamma subunit, the Gbeta5-RGS complexes isolated from native sources do not contain Ggamma. Additionally,, Ggamma is not required for the reconstitution of recombinant Gbeta 5 and RGS in vitro. Instead, Gbeta5 binds to a domain in the RGS molecule which has a striking structural homology to the Ggamma subunits. This Ggamma-like domain is present in RGS6, RGS7, RGS9 and EGL-10, an RGS from C. elegans. Since Gbeta5 as well as the RGS's, 6, 7 and 9 are predominantly expressed in the CNS, this mechanism appears to be specific for signaling in neurons. The proposed research will study the function of the Gbeta5-RGS interaction, its specificity and structural basis. AIM1 will delineate the basic molecular events that occur upon the interaction between Gbeta5, RGS and Galpha using the biochemical analysis of the proteins reconstituted in vitro. AIM2 will determine which particular Gbeta5-RGS- Galpha complexes can be found in vivo using immunological techniques. It will also characterize the multiple isoforms of Galpha, RGS and Gbeta and determine the sites of Gbeta and RGS involved in the binding using mutational analysis. AIM3 will pursue the understanding of the physiologic processed affected by Gbeta5-RGS interaction and the identification of specific receptors that signal through this pathway. These experiments will help us understand how RGS proteins control the signaling in neurons. In addition, this research is important for the development of novel therapeutics which act as regulators of RGS function. Blocking the RGS-Gbeta5 interaction, for instance by synthetic peptide that mimics the binding sites, is expected to activate the RGS and lead to inhibition of a specific signaling circuit. The basic insights and the assays developed by this research might help to design small compounds acting by this mechanism.